The cellular and KSHV A-to-I RNA editome in primary effusion lymphoma and its role in the viral lifecycle

Adenosine-to-inosine RNA editing is a major contributor to transcriptome diversity in animals with far-reaching biological consequences. Kaposi’s sarcoma-associated herpesvirus (KSHV) is the etiological agent of several human malignancies including primary effusion lymphoma (PEL). The extent of RNA editing within the KSHV transcriptome is unclear as is its contribution to the viral lifecycle. Here, we leverage a combination of biochemical and genomic approaches to determine the RNA editing landscape in host- and KSHV transcriptomes during both latent and lytic replication in PEL. Analysis of RNA editomes reveals it is dynamic, with increased editing upon reactivation and the potential to deregulate pathways critical for latency and tumorigenesis. In addition, we identify conserved RNA editing events within a viral microRNA and discover their role in miRNA biogenesis as well as viral infection. Together, these results describe the editome of PEL cells as well as a critical role for A-to-I editing in the KSHV lifecycle.

• In Fig 5d, the authors show that editing within the miRNA-K12-4-3p seed region impacts viral transmission. However, whether this editing is required by KSHV for transmission still needs to be further addressed. The authors previously describe how the pri-miRNA K12-4-3p is edited in the lower stem loop and this significantly decreases mature miRNA K12-4-3p expression in an antiviral fashion. Following miRNA processing, primary miRNA editing should occur before the editing of the mature miRNA seed region, which appears to be pro-viral. The authors could include wt and edited pri-miRNA-K12-4 in their experiments to assess whether the editing of the cleaved miRNA-K12-4 is truly required for viral transmission and the findings are not artificial. On this note, the authors should also address which type of editing is preferred within the primary vs mature miRNA transcript in either latent or lytic stage of replication. Since ADAR1 p110 is in the nucleus, it is likely the predominant editor of pri-miRNA-K12-4, while p150 may be the predominant editor of miRNA-K12-4.
• In order to discreetly analyze the impact of A-to-I RNA editing in KSHV reactivation (latent vs lytic cycle), it would be important to include the comparison of global A-I editing sites, efficiency, and its transcriptional implication between cells that were induced for lytic cycle but were GFPcells (bystander cells) within the same population. This analysis might provide insights of the mechanism of "spontaneous" lytic reactivation.
• The percentage of editing (peak height of G/(G+A)*100) in sanger sequencing could give a more accurate representation of the population of mRNA edited. This is particularly necessary for the miRNA editing observed (Fig 5d), where there seems to be lower editing compared to other mRNA regions (Fig 1h), which the authors need to address.
• The authors should focus on the novelty of their global analysis finding, further discussing specific host and KSHV transcript remodeling between latency vs lytic reactivation in addition to miRNA K12-3-4p editing sites and less emphasis on increased numeric values upon lytic reactivation. Gandy et al had previously reported that ADAR1 A-to-I editing controls the function of the Kaposin A/mi-K10 portion of the K12 transcript in PEL cells and that editing increased 10-fold by activation of lytic replication.2 On this note, Zhang et al. also showed that ADAR1 facilitates KSHV lytic reactivation by modulating the RLR-dependent signaling pathway.3 The field also knows that ADAR1 preferentially edits intronic and untranslated regions mainly in host Alu elements.1,4 • Lines 371-385, the authors discuss the potential of editing in protein coding regions. However, this study showcases how most host editing occurred in intronic (>60%) and untranslated regions (~30%) and focus on non-coding KSHV miRNA. The authors showed Kaposin and ORF50 (Fig 3c) editing in BCBL-1 cells, but this requires further follow up studies to prove adaptive RNA editing. On this note, unlike host analyses ( Fig 1d), the authors did not detail the percent editing in different types of KSHV RNA, which could strengthen their interpretations and add to their findings.
Minor critics • Line 54, as per the Wiley citation, it is small nucleolar RNA (snoRNA) not snRNA and the authors mention they promote 2'O-methlation of adenosine which impairs editing. As per the Nishikura citation, the authors report that adar1p110 accumulates in the nucleolus proposed to binding to rRNA or to small nucleolar RNA. • Line 59, ADAR1 KO mice not only have reduced adar1 editing, but the phenotype is also lethal, mainly through MDA5-mediated mechanism • Line 358, it has been reported greater Z-RNA repertoire allow p150 rather than p110 to bind to more RNA and have increased activity.5 • Line 380, please further describe and cite what is meant by adaptive RNA editing • Line 408, authors mention five PEL lines tested for conserved editing sites in pri-miR-K12-4 yet only four PEL lines are mentioned in line 244: BC-1, BC-3, BC-5, and JSC-1. • F1 b, may be unnecessary • F1 d, donut charts are simple to follow but the order and arrows into protein coding and noncoding regions needs to be simpler to follow. They also have a subset between latent and lytic which makes it more complicated.
• F1 e-f, can be supplemental • Fig 1h, there needs to be an average percent editing based on peak height • Fig 1b, the text (lines 103-104) describes how we can compare cDNA sequence and reference genomic sequence to identify A-to-I RNA editing sites. However, figure 1B shows how adenosine is converted to inosine at the RNA level. The text and figure do not match well. • Fig 1d,  Reviewer #2: Remarks to the Author: In this paper, the authors explore the roles of A-to-I editing in KSHV-infected cells. They first catalog A-to-I editing within latent and lytic cells and show that the A-to-I editome changes between phases. They examine both host and viral gene targets and then delve more deeply into the effects of KSHV miRNA editing on processing and function. Overall, the data are clearly presented and interpreted appropriately. The findings impact both the KSHV and RNA editing fields, so it has a broad audience. I have only minor comments for improvement of the manuscript: 1. Line 97: "We collected latent and lytic GFP + cells". This reads as if latent cells are GFP positive. Please clarify this sentence in the text. 2. Please label the two graphs in Figure S1c Fig 4k, they state that they "cloned the 3UTR of six predicted targets…". How are they defining "predicted" here? Does this mean predicted to be targeted by both, edited, or unedited miRNAs? If they are specific, do the results correlate with predictions? 8. The authors correctly state that the data in Fig 5c for the complementation with "miRNA with G" are not statistically significant. However, 2 replicates are quite a bit higher than the third and the lower mean and higher error is due to the third one replicate. This may point to biological relevance, particularly in light of the results in Fig 4d. How confident are they that the lytic replication is not better in these cells? This should perhaps be noted. 9. Overall, the labels on figures tend to be too small.
Reviewer #3: Remarks to the Author: This manuscript reports the A to I editing patterns of cells infected with KSHV virus, and in either the latent or reactivating state of infection. They demonstrate that there is a substantial increase in A-I editing of the KSHV genome and of the cellular genome in cells undergoing lytic reactivation, and that the A-I editing pattern in these 2 states of infection has some overlap, but also has very clear distinctions in the A-I edited sites. These findings are exciting and novel, and provide insights into an important source of RNA editing during infection, with potential for biological outcomes during infection. They go on to show that one of the edited sites in a viral miRNA results in a functional change in the miRNA, such that the result is a significant decrease in virus lytic reactivation. These new insights are strong and are important to the fields of RNA biology and to virus-host interactions. The major conclusions and large datasets reported in this manuscript (above) are sound, yet there are some secondary conclusions and details that are not strong enough to support the conclusions. Particularly, the authors have not demonstrated that modification of the miRNA-K-12-4 sequence results in downstream defects in virus binding and entry. Major concerns: 1) Several experiments use transfected/modified cells that are not well-described and for which there is no validation data provided. In nearly every case, the pedigree and timing of the cells should be clarified for the reader, potentially by timeline schematics or tables. a) the pPAN reporter cells are very briefly described, and are essential to the study. Data should demonstrate their inducibility relative to parent lines and their GFP status with and without induction. Evidence that the sorts result in pure populations of latent vs lytic reactivating cells could be further supported by the RNA Seq files. Flow cytometry data should include stained and unstained samples, controls, live/dead discrimination, gating strategies, and comparison of presort and post-sorted cells. Clarification on whether 'latent cells' refer to untreated cells or cells sorted and selected as GFP-negative. The same goes for the PAN fish flow staining, PAN anti-sense oligos should be listed, and methods are unclear on which Alexafluor is used in experiments shown. b) the iSLK and BCBL-TRex cell transfection schemes should be clarified. There are a number of miRNA and mimics and each could be made more plain for the reader. Text and Methods differ in transfection methods. 2) While the manuscript does a very convincing demonstration of A-I editing distinctions and shows quite well that A-I editing of a viral miRNA compromised virus lytic reactivation, there are subsequent studies of the resulting virus that are not well supported. In Figure 5d, the authors show that miRNA-K12-4 mimics with sequences matching the edited or unedited sequences result in striking differences in virus reactivation with the result of significantly decreased virus production. However, in Figure 5f-h, the data shown is taken to mean that the resulting virus from this supernatant is itself defective in subsequent binding and entry. If this were to be proven, it would require inoculation of equivalent amounts of virus for analysis of binding and entry. This is a significant burden of proof, and could be quite difficult given the very poor virus production shown in Figure 5c/d. Methods state that these cells were infected with an MOI=3 particles, but no particle measurements can be made without EM or Virocyte, so it is unclear what is meant here. Viral DNA is reported as relative to WT rather than relative to input, and no limit of detection or background level is provided. As is presented, there is not convincing data shown to refute the reasonable conclusion that reduced virus production results in reduced virus available for binding and entry.
3) The schematic refers to virus infection as transmission, however, virus transmission is the process by which viruses spread between hosts. MINOR points: 1) Figure 2h, there is some H3 signal in the cytoplasmic fractions. Comment on whether this relates to virus infection and nuclear membrane integrity? 2) Figure 2j/k: comment on relatively rare events beyond 2-fold changed? 3) Figure 4e has an unlabeled white box below "KSHV miRNA vectore with G", and Figure 4e has a floating "0.0" below the miRNA-K12-9 label.
We appreciate the reviewers taking time to provide us with comments. Below we provide a point by point response. Appropiate changes have also been added to our manuscript.

Reviewer #1
The manuscript by Rajendren et al. provides the first global analysis of the A-to-I editome in PEL cells and KSHV transcriptome during KSHV latent and lytic replication. Using modified PEL cell lines, the authors collected latent and lytic GFP+ cells for deep RNA sequencing analysis to identify A-to-I edited sites using the SAILOR algorithm and further validated editing of specific sites by comparing the cDNA with genomic DNA through Sanger sequencing. Excitingly, the authors identified conserved and stage-specific A-to-I editing sites in the transcriptome of host BCBL1 and BC-3 cells and KSHV during latent and lytic reactivation. The authors further identified three previously unknown ADAR1-mediated conserved editing sites of the KSHV pri-miRNA-K12-4 transcript, two in the lower stem region and one within the seed region of mature miRNA-K12-4-3p, which affect (1) miRNA biogenesis, (2) target specificity, and (3) viral transmission. Using an established KSHV miRNA vector, the authors mutated the adenosines into guanosines within the lower stem region of pri-miRNA-K12-4 and showed a (1) significant decrease in mature miR-K12-4-3p and -5p not observed upon ADAR1 siRNA-mediated depletion. Using bioinformatics tools and luciferase assays, the authors showed that (2) editing of the seed sequence of miRNA-K12-4-3p expanded target recognition. Using the iSLK-BAC16 KSHV infection cell model with rescued edited miR-K12-4, the authors showed that (3) editing at the seed region increases infectious virion production. These findings shed light into the functional and biological significance of A-to-I editing in KSHV latent and lytic replication in PEL cells, opening the door for future applications. However, main concerns are the lack of ADAR1 isoform detection that may impact data interpretation, the importance of seed region editing of mature miRNA-K12-4 for KSHV transmission, lack of in-depth discussion of host and KSHV transcriptome remodeling during viral stages, and lack of percent editing in the Sanger trace to showcase the population of RNA edited.
Additional experimentation is required to support the reported findings.

Comment: The authors do not to take into consideration the reported isoforms of ADARBased on reported literature, it is likely that in latent infection, p110 is the main isoform which is predominantly found in the nucleus and correlates with Fig 2h ADAR1
nuclear localization. Upon lytic reactivation, the IFN-inducible p150 isoform may be expressed which localizes into the cytosol and has been reported to edit more substrates and more sites than p110. The increased editing activity the authors observed may not only be due to increased gene expression but also induction of p150 isoform. The authors need to address both isoforms and show their expression in respective western blots. In lines 366-370, the authors discussed the slight localization of ADAR1 (i.e., p110) known to shuttle between the nucleus and cytosol upon stress (Fig  2h), but neglect the major cytosolic isoform, p150.

Response:
The antibody that we used can detect both the p110 and p150 isoforms. Similar to the recent data from the Damania group (PMCID: PMC7319254), we do not observe induction of the p150 ADAR1 isoform during lytic reactivation. We have now added to lines 183 -184 indicating that we do not observe changes in the expression of the p150 ADAR1 isoform and that this is consistent with a previous report. This is also further noted on lines 389 -391.
Furthermore, our data in Figure 2i supports that the editing increase is mediated by the p110 ADAR1 isoform as the p32 CLIP signal migrates between 100kDa and 130kDa, and no p150 p32 signal is observed. Fig 5d, (Fig S2). We can detect editing at all three sites in both nuclear and cytoplasmic fractions, suggesting KSHV miRNA editing occurs within nucleus. Moreover, quantification of editing from the Sanger results indicates there is no additional editing of the KSHV miRNA occurring in the cytoplasm and our quantification from latent and lytic infected cells suggests no significant changes in the editing levels in lytic reactivation.

Comment: In
The experiment suggesting to include wt and edited pri-miRNA is not feasible for multiple reasons, including that transfection of a full-length pri-miRNA is likely trip to cell intrinsic double stranded (ds)RNA sensors and activate an interferon response confounding any results. Additionally, transfection of a wt pri-miRNA would still be edited by endogenous ADAR. Our data in Fig 5 clearly show that loss of miR-K12-4 results in the production of virus that is severally attenuated in terms of its ability to establish a latent infection, and that only an edited miRNA is capable of rescuing.

Comment: In order to discreetly analyze the impact of A-to-I RNA editing in KSHV reactivation (latent vs lytic cycle), it would be important to include the comparison of global A-I editing sites, efficiency, and its transcriptional implication between cells that were induced for lytic cycle but were GFP-cells (bystander cells) within the same population. This analysis might provide insights of the mechanism of "spontaneous" lytic reactivation.
Response: Thank you for this interesting comment. This study is focused on defining how latent (resting) and lytic editomes are remodeled and the biological impact of that editing on infection. How the GFP-(bystander cells) respond to lytic infection is a fascinating question, not only in terms of the editome but general biology. At present, those studies are out of the scope of this manuscript. However, part of our longer-term efforts towards investigating A-to-I editing plan to investigate "bystander cell" responses.

Comment:
The percentage of editing (peak height of G/(G+A)*100) in sanger sequencing could give a more accurate representation of the population of mRNA edited. This is particularly necessary for the miRNA editing observed (Fig 5d), where there seems to be lower editing compared to other mRNA regions (Fig 1h), which the authors need to address.
Response: Thank you for this comment. We believe the reviewer is refereeing to Fig 4b  and 4c instead of 5d, as there is no Sanger sequencing in 5d. The stoichiometry of RNA modifications is an important topic in the RNA modification field and can vary from very low to essentially 100%. In response to this comment, we now report the percentage of editing for all our Sanger sequencing analysis in Supplementary

Comment: The authors should focus on the novelty of their global analysis finding, further discussing specific host and KSHV transcript remodeling between latency vs lytic reactivation in addition to miRNA K12-3-4p editing sites and less emphasis on increased numeric values upon lytic reactivation. Gandy et al had previously reported that ADAR1 A-to-I editing controls the function of the Kaposin A/mi-K10 portion of the K12 transcript in PEL cells and that editing increased 10-fold by activation of lytic replication.2 On this note, Zhang et al. also showed that ADAR1 facilitates KSHV lytic reactivation by modulating the RLR-dependent signaling pathway.3 The field also knows that ADAR1 preferentially edits intronic and untranslated regions mainly in host Alu elements.1,4
Response: We thank the reviewer for this comment. As this is the first comprehensive analysis of host and viral editing in PEL and the KSHV lifecycle we believe it is important to include the global quantitative view of editing. Particularly, as the data here can serve as a great resource for future investigations into the biology of A-to-I editing during KSHV infection.
Thank you for pointing out these references. In fact, in our previous manuscript all of these were cited. However, we now include additional comments for each citation highlighting points made by the reviewer (lines 236, 441-442, and 443-445). (Fig 3c) editing in BCBL-1 cells, but this requires further follow up studies to prove adaptive RNA editing. On this note, unlike host analyses ( Fig 1d), the authors did not detail the percent editing in different types of KSHV RNA, which could strengthen their interpretations and add to their findings.

Response:
Our studies provide a deep and comprehensive analysis of the A-to-I RNA editing landscape in PEL and the effect of editing on KSHV miRNA biology. The potential for RNA editing to influence the proteome is well established, both in humans and in other organisms, and we agree that if investigators seek to test the effect of protein recoding on the KSHV lifecycle additional studies will be required. Indeed, while proteome recoding is clearly out of the scope of this manuscript, it is a topic that we are actively investigating. In Fig 4a, please graphically depict the sequence of the mature miRNAs and seed sequences on the pri-miRNA. It would be helpful for readers to know where these features are to compare them to the sites of editing.

Response:
We thank the reviewer for this comment. Mature miRNA sequences are graphically depicted in our revised pri-miRNA-K12-4 schematic with seed sequences of both 3p and 5p miRNAs marked in addition to the edited sites.
6. Comment: In Fig 4d the

Response:
We have now repeated these experiments in triplicate and observe less variation in the new data. The new data is included in the revised Fig 5C and suggests that the number of viral genomes in the supernatant collected from the complementation experiment is not significantly different between our five samples.

Comment:
Overall, the labels on figures tend to be too small.

Response:
The size of the labels has been adjusted.

Reviewer #3:
This manuscript reports the A to I editing patterns of cells infected with KSHV virus, and in either the latent or reactivating state of infection. They demonstrate that there is a substantial increase in A-I editing of the KSHV genome and of the cellular genome in cells undergoing lytic reactivation, and that the A-I editing pattern in these 2 states of infection has some overlap, but also has very clear distinctions in the A-I edited sites. These findings are exciting and novel, and provide insights into an important source of RNA editing during infection, with potential for biological outcomes during infection. They go on to show that one of the edited sites in a viral miRNA results in a functional change in the miRNA, such that the result is a significant decrease in virus lytic reactivation. These new insights are strong and are important to the fields of RNA biology and to virus-host interactions. The major conclusions and large datasets reported in this manuscript (above) are sound, yet there are some secondary conclusions and details that are not strong enough to support the conclusions. Particularly, the authors have not demonstrated that modification of the miRNA-K-12-4 sequence results in downstream defects in virus binding and entry. Response: Thank you for this comment. We added more details to the iSLK and TRex-BCBL1 transfection schemes for clarification.

Comment: While the manuscript does a very convincing demonstration of A-I editing distinctions and shows quite well that A-I editing of a viral miRNA compromised virus
lytic reactivation, there are subsequent studies of the resulting virus that are not well supported. In Figure 5d, the authors show that miRNA-K12-4 mimics with sequences matching the edited or unedited sequences result in striking differences in virus reactivation with the result of significantly decreased virus production. However, in Figure 5f-h, the data shown is taken to mean that the resulting virus from this supernatant is itself defective in subsequent binding and entry. If this were to be proven, it would require inoculation of equivalent amounts of virus for analysis of binding and entry. This is a significant burden of proof, and could be quite difficult given the very poor virus production shown in Figure 5c/d. Methods state that these cells were infected with an MOI=3 particles, but no particle measurements can be made without EM or Virocyte, so it is unclear what is meant here. Viral DNA is reported as relative to WT rather than relative to input, and no limit of detection or background level is provided. As is presented, there is not convincing data shown to refute the reasonable conclusion that reduced virus production results in reduced virus available for binding and entry.

Response:
We apologize for the confusion as there is some misinterpretation in the way we presented panels in Fig 5. To aid in clarification we have modified Fig 5 and corrected (i.e.) removed particles from MOI as it was a typo.
In Figure 5b and 5c we demonstrate that viral reactivation (5b, gene expression; 5c virion production/genome numbers) are not affected by loss of miR-K12-4. We now clearly indicated 5b is lytic gene expression, and 5c is now viral genomes/mL. In Figure 5d, which is the supernatant transfer assay, loss of miR-K12-4 results in a significant reduction in infectious virions, that can only be rescued by providing the edited miR-K12-4. We now modify the schematic to indicate it is the supernatant transfer. To investigate virus binding and entry we performed assay's similar to those previously described in (PMCID: PMC3894220), in which we used a similar number of viral genomes for binding and entry assays.

Comment: The schematic refers to virus infection as transmission, however, virus transmission is the process by which viruses spread between hosts.
Response: Thank you for this comment. We used transmission as we were transmiting the virions produced from iSLK-bac16 cells to another cell line (either HEK 293T or HUVECs). However, we have now replaced 'transmission' with 'infection' in the revised manuscript. Reviewers' Comments: Reviewer #1: Remarks to the Author: In this revision, the authors defined the ADAR1 isoform involved, ADAR1 p110, and included a report of the editing percentage of their Sanger sequencing analyses in addition to SAILOR quantification of both host and viral edited sites. The authors also addressed their longer-term efforts to investigate GFP-bystander cell responses, included the multiple references to be added, and addressed the setbacks of attempting to annotate the KSHV editing genome and incorporating edited pri-miRNA. The authors incorporated all minor comments, including figure modifications, labeling, figure description as well as additional points of discussion, references, and necessary replicates. Overall, the authors have addressed all major comments and their additional experimentation and revision improved their original submitted manuscript. I only have additional minor comments. Minor comments • Figure 1h and Figure 4b. The Sanger trace seem as if they were the same figure repeated for the different cell lines genomic cDNA traces for one target. If the figures are just a representation of genomic cDNA target, including only one can make interpretations clearer.
• Figure 4L. Legend for dark blue is missing. Addition of FigS3B provides critical support for the validity of the cells and sorts-great! Figure 5H still refers to "transmission" which should be changed or tempered because it is not proven. However, the idea that ADAR editing could impact transmission is interesting, and could be included in discussion on possibilities. The revised manuscript now referenced "MOI=3", but should include units (3 what?) such as genome equivalents or what best describes virus quantitation here.

Reviewer #1
In this revision, the authors defined the ADAR1 isoform involved, ADAR1 p110, and included a report of the editing percentage of their Sanger sequencing analyses in addition to SAILOR quantification of both host and viral edited sites. The authors also addressed their longer-term efforts to investigate GFP-bystander cell responses, included the multiple references to be added, and addressed the setbacks of attempting to annotate the KSHV editing genome and incorporating edited pri-miRNA. The authors incorporated all minor comments, including figure modifications, labeling, figure description as well as additional points of discussion, references, and necessary replicates. Overall, the authors have addressed all major comments and their additional experimentation and revision improved their original submitted manuscript. I only have additional minor comments.
Comment: Figure 1h and Figure 4b. The Sanger trace seem as if they were the same figure repeated for the different cell lines genomic cDNA traces for one target. If the figures are just a representation of genomic cDNA target, including only one can make interpretations clearer.
Response: Thank you for this comment and we really appreciate it. After careful consideration, we still want to report the genomic DNA traces from every cell line we assessed the editing sites to show the reader that the genomic traces are purely adenosines at the edited sites. This will eliminate any concern the reader might have on whether the genomic DNA from different cell lines contains SNPs.
Comment: Figure 4L. Legend for dark blue is missing.

Response:
We thank the reviewer for this comment and the legend was added.

Reviewer #3
This manuscript regarding ADAR editing patterns of KSHV infected cells, in either latent (untreated) or reactivating state of infection. They demonstrate that there is a substantial increase of A-I editing of both KSHV and the cellular genomes in cells undergoing lytic reactivation, and demonstrate distinct edited sites. These findings are exciting and novel and provide insights into an important source of RNA editing during infection. This revised manuscript has addressed the majority of reviewer concerns, however, there remain outstanding details. These are critical to reader understanding and to the potential for others to replicate the study or to implement methods or reagents. These are essential parts of any publication and can be easily revised to meet standards for rigor and authentication.